Leaf Spot Caused by Irpex lacteus on Tobacco in China

Tobacco (Nicotiana tabacum L.) is an important commercial crop in China. In June 2023, disease symptoms were seen in a commercial tobacco field in Bijie City (27.09° N, 105.36° E), Guizhou province, China. Typical symptoms first appeared on the lower leaves as round brown spots, with a light center and a faint yellow small halo. Approximately 25% of the plants were symptomatic in a 3.5-ha field. From 8 plants, 16 small pieces of leaf tissue (6 × 6 mm) were cut from the edge of the lesions, including live and dead portions. These were surface sterilized in 1% hypochlorite for 30 s, and then placed on potato dextrose agar (PDA) amended with kanamycin (0.1 mg/ml). After 48 hours at 28 °C, 12 fungal strains were recovered. Eight strains presented a consistent morphology, while the remaining four strains varied in morphology. All 12 strains were tested for pathogenicity on detached leaves and only the eight (presenting a consistent morphology) showed disease. The other four strains showed no disease. A representative isolate, MDY, was selected for causal agent identification. The hyphae were white, slightly raised, thin-walled, straight, and 2.0-4.0 μm in diameter. There was no detectable odor. Conidia were numerous, clustered, ovoid, colorless, and 5-8 µm x 4-5 µm. These characteristics were similar to those reported for Irpex lacteus (Chi et al. 2002). The internal transcribed spacer (ITS), large ribosomal subunit (LSU) and small ribosomal subunit (SSU) loci of isolate MDY were, respectively, amplified with primers ITS1/ITS4, LR5/LROR, and NS1/NS4 (Schoch et al. 1998; Melnikov et al. 2012), and sequenced using both forward and reverse primers. Consensus sequences from each pair of primers were used in BLAST against the NCBI GenBank database. The ITS sequence (OR366346) showed 99.9% identity (648/649 bp) with I. lacteus (MH890689), the LSU sequence (OR878461) showed 99.6% identity (904/908 bp) with I. lacteus (MH867969), and the SSU sequence (OR878462) showed 99.5% identity (969/974 bp) with I. lacteus (MF190370). Neighbor-Joining analysis was carried out using the ITS sequence of MDY, several related sequences from GenBank, and Macrocybe gigantea OQ644634 as outgroup. MDY clustered with other I. lacteus sequences with 95% bootstrap support. Morphological and molecular results supported this isolate as I. lacteus. Pathogenicity was tested on six tobacco seedlings (cv. Yunyan 87) at the six-leaf stage using hyphal plugs from isolate MDY on non-wounded attached leaves, while controls received PDA plugs. This test involving three treated plants and three control plants was repeated three times. All tobacco seedlings were kept in a growth chamber at 25 ± 5 °C and 85% relative humidity. After 7 days, leaf spots similar to field symptoms were observed on the treated leaves, while no symptoms were observed on control leaves. Koch's postulates were fulfilled by re-isolation of the pathogen from the diseased leaves, confirmed by ITS sequencing. From previous work, I. lacteus was described and reported as an endophyte in tobacco in Enshi, Hubei (Yuan et al. 2018), and some endophytes are known to be latent pathogens (Brown et al. 1998), but to the best of our knowledge, this is the first report of I. lacteus causing tobacco leaf spots. Due to potential serious damage caused by this disease on tobacco in China, further research is needed to establish its incidence in tobacco fields and the source of the inoculum in localized infections.